Assays that rapidly and efficiently identify cells that have been successfully transformed with exogenous DNA are useful in a wide variety of applications including, for example, identifying cell lines and/or transgenic organisms and conducting high-throughput drug screening. Indeed, it is only once the insertion of the exogenous DNA is confirmed that cell lines or transgenic organisms can be used as animal models for drug discovery, elucidation of biochemical pathways, toxicity studies and the like.
The most commonly used method for detecting whether exogenous nucleic acid has been introduced into a target cell is by selection, either by a nutritional deficiency that is complemented by the introduced DNA, or by drug resistance. For complementation of auxotrophic mutants, the recipient cells have to be deficient in that gene function. For drug resistance, the cells have to be sensitive to that drug. Unfortunately, many cells (e.g., pathogens) are resistant to drug selection techniques. Candida albicans, for example, displays resistance to hygromycin, benomyl, cycloheximide, mitomycin C and tunicamycin. Similarly, other pathogenic organisms (e.g., methicillin-resistant Staphylococcus aureus (MRSA), and some variants of M. tuberculosis) have developed resistance to many of these routinely used antibiotics. Thus, it has proven very difficult to transform these cells and to detect successful integration events.
Methicillin-resistant strains of Staphylococcus aureus (MRSA) are the most common nosocomial pathogens worldwide. MRSA are responsible for greater than 40% of hospital-born staphylococcal infections in large, U.S., teaching hospitals. They have become prevalent in smaller hospitals (20% incidence in hospitals with 200 to 500 beds), as well as in nursing homes (Wenzel et al., 1992, Am. J. Med. 91(Supp 3B):221-227). An unusual property of MRSA strains is their ability to pick up additional resistance factors resulting in little or no susceptibility of these strains to chemotherapeutically useful antibiotics. Such multi-resistant strains of bacteria are now prevalent world-wide and the worst of these pathogens carry resistance mechanisms to all but one (i.e., vancomycin) of the usable antibacterial agents (Blumberg et al., 1991, J. Inf. Disease (63:1279-1285).
Another nosocomial pathogen, Enterococcus faecium, is known for its ability to transfer from one cell to another plasmid-born resistance factors, such as, vancomycin resistance. Such a mechanism for the transfer of resistance factors will lead to more and more bacterial resistant to known antibiotics. High-level vancomycin-resistance strains of E. faecium were first isolated in England in 1986. In 1990s, Vancomycin-resistant enterococci (VRE) were documented as having been spread all over the world. The identification of multidrug-resistant strains, which are resistant to high concentrations of ampicillin or gentamicin, or to vancomycin, introduces serious therapeutic dilemmas. Further, the absence of sensitivity to antibiotics greatly interferes with the ability to manipulate these organisms, particularly in view of the fact that most bacterial transformation vectors rely on antibiotic resistant selection. A more complete explanation of the basis for antibiotic resistance and the emergence of resistant strains can be found in the literature (e.g., U.S. Pat. No. 6,136,587 Tomasz, et al. Oct. 24, 2000; Ohno, A., et al., Nippon Rinsho (2001) 59(4):673-680).
The lack of efficient screening systems in certain organisms can seriously impair efforts to screen candidate drugs. In part due to the increasing numbers of immunocompromised patients, infections with drug-resistant organisms and systemic fungal infections with normally benign organisms are on the rise, for example, many such infections are due to the normal human flora Candida albicans. Candida species are now the fourth most common cause of nosocomial bloodstream infections (Edmond et al. (1999) Clin Infect Dis. 29(2):239-44). Commensurate with the increase in the number of people contracting serious Candida infections, is an increase in the incidence of strains resistant to antifungal compounds (White, Marr, et al., (1998) Clin Microbiol Rev 11(2):382-402). To counteract this, new classes of antifungal compounds and their possible targets must be investigated.
The study in Candida albicans of new targets amenable to antifungal attack, using genetic approaches, has been hampered by several factors. First, the plasmids used with Candida albicans are usually present in cells as tandem copies and are quickly lost without selective pressure (Cannon et al. (1992) Mol Gen Genet. 235(2-3):453-457; Kurtz et al. (1987) Mol Cell Biol. 7(1):209-17; Pla et al. (1995) Gene 165(1):115-20). Further, unlike Saccharomyces cerevisiae, no centromeric sequence or autonomously replicating sequence has been cloned which would allow episomal plasmids to be maintained without any selection.
Another factor that has hampered the study of C. albicans and the generation of effective treatments that this organism was thought to be diploid throughout its life cycle. Only recently has it been shown that Candida can be forced to mate (Magee (2000) Science 289(5477):310-313; Hull et al. (2000) Science 289(5477):307-310), possibly going through a tetraploid state. The presence of genetic elements akin to the mating alleles of S. cerevisiae suggests that meiotic division may lead to haploid cells, however such haploid isolates have not been observed. Accordingly, to study a new antifungal target, both chromosomal copies of the candidate gene must be inactivated see its effect on the cell. To knock out both copies, a construct called a “ura blaster” was developed (Alani (1987) Genetics 117(1):5-12; Fonzi et al. (1993) Genetics 134(3):717-28). Cells must be made auxotrophic for URA3, and creating an auxotrophic strain from a clinical isolate usually reduces virulence significantly or totally abolishes it (Lay et al. (1998) Infect Immun. 66(11):5301-6; Kirsch et al. (1991) Infect Immun. 59(9):3297-300; Polak (1992) Mycoses 35(1-2):9-16; Cole (1995) FEMS Microbiol Lett. 126(2):177-80). A study has shown that even in virulent strains, recovery of URA3 function may not necessarily restore virulence to wild type levels (Lay et al. (1998), supra).
A third issue is that very few heterologous genes have been expressed in C. albicans due to its abnormal codon usage (CTG coding for serine instead of leucine) (Leuker (1994) Mol Gen Genet. 245(2):212-7). In order for a heterologous gene to be functionally expressed in Candida albicans, every CTG codon in the gene must be mutated to other leucine codons (Morschhauser et al. (1998) Mol Gen Genet. 257(4):412-20). Thus, these and other difficulties have limited the study of Candida genes to strains in which URA3 or another nutritional marker has been knocked out. Many reporter genes are not used due to the effort needed to mutagenize them for expression.
Methods described in U.S. Pat. No. 5,650,135, make possible the detection of bioluminescent bacteria in a living animal without dissecting or otherwise opening the animal up (“in vivo monitoring”)—the light is detected through muscle, skin, fur & other traditionally “opaque” tissues using a highly sensitive camera. Although green fluorescent protein (GFP) has been expressed in C. albicans (Cormack, Bertram, et al., (1997) Microbiology 143(Pt 2):303-11; Morschhauser, Michel, et al., (1998) Mol Gen Genet 257(4):412-20), GFP producing C. albicans cells have not yet been imaged inside a living animal. Srikantha, et al., ((1996) J Bacteriol. 178(1):121-9) reported expression of the luciferase of the sea pansy Renilla reniformis in C. albicans, but the cost of the substrate, coelentrazine, makes it impractical for use in living animals.
In addition to C. albicans many other pathogenic organisms have become resistant to most or all antibiotics, thus making transformation of these organisms difficult, if not impossible by classical methods of introducing DNA comprising a drug resistance gene and selecting for drug resistance conferred by the DNA used for transformation.
Thus, there remains a need for methods of detecting transformation of target cells, particularly methods that do not involve selectable markers. The present invention provides, inter alia, such methods, expression cassettes, transposon cassettes and other tools useful for generating light-producing organisms, for example, pathogenic organisms (such as, MRSA, antibiotic-resistant bacteria, and Candida albicans) which are suitable for studies relating to infection and/or pathogenesis in vitro and in whole animals.